The present invention relates to polypeptides, polynucleotides and uses thereof in screening and in medicine.
Obesity is a serious health hazard that may be accompanied by elevated levels of blood cholesterol and elevated blood pressure. There is an increased risk of mortality from coronary heart disease, stroke, certain cancers and non-insulin dependent diabetes, as well as an association with several non-fatal health problems.
Obesity is usually measured by Body Mass Index (BMI), which is obtained from a simple formula, given by:
BMI=(mass/kg)xc3x97(height/m)xe2x88x922
BMI values in adults have been categorised according to the severity of health risk, with the higher graded numbers associated with a more severe risk (Table 1). Modified versions of this scale have been derived which account for gender, and there are other considerations such as age and body frame (see, for example Black""s Medical Dictionary).
Desirable body weights for adults according to gender, height and build are given in Appendix 6C of Black""s Medical Dictionary.
Ischia et al (1997) J Biol Chem 272, 11657-11662 describes the cloning of a bovine chromogranin-like polypeptide termed NESP55 (neuroendocrine secretory protein of Mol Wt 55,000). A partial sequence for mouse NESP55 is also presented. As reviewed by Ischia et al, chromogranins are proteins found in the content of large dense core vesicles, specialised vesicular containers found in presynaptic terminals which store neurotransmitters and peptides prior to exocytic release during synaptic transmission. Chromogranins have an acid pI of 4 to 5 and typically consist of 200-700 amino acids with glutamic acid as the most abundant individual amino acid. Multiple pairs of consecutive basic amino acid residues, known as potential cleavage sites for trypsin-like endoproteases, are present. Both intracellular (involvement in sorting of peptidergic components to the large dense core vesicles) as well as extracellular functions (representing precursors of small biologically active neuropeptides such as pancreastatin, vasostatin or secretoneurin) have been proposed for chromogranins.
Bovine NESP55 is proposed to be a precursor for the tetrapeptide Leu-Ser-Ala-Leu (SEQ ID NO: 3), which has been identified as an endogenous antagonist of the serotonergic 5-HT1B receptor subtype. The serotonergic system is thought to play a role in mental disorders, particularly depression. A second amino acid sequence, GAIPIRRH (SEQ ID NO: 4), present at the C-terminus of bovine NESP55 is the same as that of a peptide identified in the secretory content of chromaffin granules (Sigafoos et al (1993) J Anat 183, 253-264). A function was not assigned to this peptide, and there has been no suggestion that either peptide is involved in obesity.
We have identified a human homologue of the bovine NESP55 gene. Further, we have found that NESP55 is, at least in humans, linked with obesity. Accordingly, the invention provides isolated human neuroendocrine secretory protein 55 (NESP55).
In one aspect, the invention provides a substantially pure polypeptide comprising the amino acid sequence of SEQ ID NO: 2 or a variant, fragment, fusion or derivative thereof, or a fusion of a said variant or fragment or derivative, wherein the polypeptide variant has an amino acid sequence which has at least 90% identity with the amino acid sequence of SEQ ID NO: 2. The invention also provides a processed polypeptide derivable from human NESP55 wherein the processed polypeptide is derivable, or predicted from the amino acid sequence of human NESP55 to be derivable, by endoproteolytic cleavage of human NESP55. In one embodiment, the N-terminal amino acid residue of the processed polypeptide is immediately preceded in the amino acid sequence of human NESP55 by two consecutive basic amino acid residues or by a basic amino acid residue. In another embodiment, the C-terminus of the processed polypeptide is immediately preceded by two consecutive basic amino acid residues or by a basic amino acid residue. In preferred embodiments, the processed polypeptide comprises the amino acid sequence LHAL (SEQ ID NO: 5) or the amino acid sequence GPIPIRRH (SEQ ID NO: 6), or consists of the amino acid sequence LHAL (SEQ ID NO: 5) or the amino acid sequence GPIPIRRH (SEQ ID NO: 6).
The invention also provides a polypeptide consisting of the amino acid sequence XnLHALZm (SEQ ID NO: 11), or XnGPIPIRRHZm (SEQ ID NO: 12) wherein Xn represents the amino acid sequence of the consecutive n amino acids immediately N terminal to the amino acid sequence LHAL (SEQ ID NO: 5) or GPIPIRRH (SEQ ID NO: 6) and wherein Zm represents the amino acid sequence of the consecutive m amino acids immediately C terminal to the amino acid sequence LHAL (SEQ ID NO: 5) or GPIPIRRH (SEQ ID NO: 6), wherein n and m may independently be any number between 0 and 30 amino acids. In one embodiment, one or both of Xn or Zm consists of the sequence immediately flanking the LHAL (SEQ ID NO: 5) or GPIPIRRH (SEQ ID NO: 6) sequences in native human NESP55.
15. The invention also provides an isolated polypeptide comprising the amino acid sequence of SEQ ID NO:2, as well as isolated polypeptides comprising an amino acid sequence at least 90% identical to the amino acid sequence of SEQ ID NO: 2, or comprising an amino acid sequence at least 95% identical to the amino acid sequence of SEQ ID NO: 2, or comprising an amino acid sequence at least 98% identical to the amino acid sequence of SEQ ID NO: 2, or comprising an amino acid sequence at least 99.5% identical to the amino acid sequence of SEQ ID NO: 2. Isolated peptides consisting of the amino acid sequence LHAL (SEQ ID NO: 5) or the amino acid sequence GPIPIRRH (SEQ ID NO: 6) are also provided.
Another aspect of the invention pertains to isolated polynucleotides encoding or complementary to a polynucleotide encoding the polypeptides of the invention, e.g. encoding human NESP55 or encoding SEQ ID NO: 2. In a preferred embodiment, the isolated polynucleotide comprises the nucleotide of SEQ ID NO: 1. In another preferred embodiment, the polynucleotide is suitable for expressing the polypeptide of the invention in a host cell. The invention further provides vector constructs comprising the polynucleotides of the invention, and host cells transformed with the vector constructs of the invention. Still further, methods of making human NESP55 polypeptide, comprising culturing a host cell of the invention and isolating the human NESP55 polypeptide from the host cell, or culture medium, are also provided. Polypeptides obtainable by these methods are also encompassed by the invention.
Yet another aspect of the invention pertains to antibodies reactive towards the polypeptides of the invention. In one embodiment, the antibody is reactive towards the peptide sequence LHAL (SEQ ID NO: 5) and GAIPIPIRRH (SEQ ID NO: 6).
Pharmaceutical compositions, comprising an antibody of the invention, a NESP55 polypeptide of the invention or a NESP55 processed polypeptide of the invention, as well as a pharmaceutically acceptable carrier, are also provided. Methods of treating or preventing obesity in a patient, the comprising administering to the patient an effective amount of an antibody of the invention, a NESP55 polypeptide of the invention, a NESP55 processed polypeptide of the invention or a pharmaceutical composition of the invention are also provided.
Another aspect of the invention pertains to a method of identifying a polypeptide (interacting polypeptide) that is capable of interacting with a human NESP55, or a processed polypeptide thereof. The method comprises the steps of (1) exposing the polypeptide to a test composition that may comprise an interacting polypeptide, (2) detecting an interaction between the polypeptide and an interacting polypeptide and optionally (3) identifying and/or isolating the interacting polypeptide. A substantially pure interacting polypeptide identified or identifiable by the method of the invention is also provided.
Another aspect of the invention pertains to a method of identifying a compound capable of disrupting or preventing the interaction between a human NESP55 polypeptide, or a processed polypeptide thereof or a GAIPIRRH (SEQ ID NO: 4)-containing polypeptide, and an interacting polypeptide. In the method, the human NESP55 polypeptide, or processed polypeptide thereof or the GAIPIRRH (SEQ ID NO: 4)-containing polypeptide, and the interacting polypeptide are exposed to the compound and the interaction between the human NESP55 polypeptide, or processed polypeptide thereof or the GAIPIRRH (SEQ ID NO: 4)-containing polypeptide, and the interacting polypeptide is measured in the presence and absence of the compound. Compounds identified by or identifiable by this method are also encompassed by the invention.
Still a further aspect of the invention pertains to a method of determining whether an individual is likely to become or remain obese or become more obese. The method comprises determining the level of NESP55 or a fragment derived or derivable from NESP55 or determining the level of a messenger RNA encoding NESP55, or the activity of NESP55 in a tissue sample, for example a body fluid, and determining that the level or activity differs from a level or activity found in an individual that is not obese and/or not expected to become obese. A kit of parts for determining whether an individual is likely to become or remain obese or become more obese the kit comprising means to determine the level of NESP55 or a fragment derived or derivable from NESP55 or means to determine the level of mRNA encoding NESP55 is also provided.
Another aspect of the invention pertains to a method of treating or preventing obesity in a patient, the method comprising administering to the patient an effective amount of an inhibitor of a polypeptide that is capable of cleaving NESP55. A compound capable of altering the expression of NESP55 is also encompassed by the invention, as well as a method of treating or preventing obesity in a patient, the method comprising administering to the patient an effective amount of a compound capable of altering the expression of NESP55.
Yet another aspect of the invention pertains to a method of identifying a compound capable of disrupting or preventing the interaction between the peptide LHAL (SEQ ID NO: 5) and human 5HT1B/1D receptor wherein the LHAL(SEQ ID NO: 5)-containing polypeptide and/or the receptor are exposed to the compound and the interaction between the polypeptide and the receptor is measured in the presence and absence of the compound. A kit of parts comprising a LHAL (SEQ ID NO: 5)-containing peptide and human 5HT1B/1D receptor is also provided. A compound identified by or identifiable by this method is also provided.
Another aspect of the invention pertains to a method of disrupting or preventing the interaction between the peptide LHAL (SEQ ID NO: 5) and human 5HT1B/1D receptor. The method comprises exposing the human 5HT1B/1D receptor to a compound identified by the above method or an antibody reactive against the peptide sequence LHAL (SEQ ID NO: 5). The invention also provides a method of treating or preventing obesity in a patient, the method comprising administering to the patient an effective amount of the compound identified by the above method or an antagonist of human 5HT1B/1D receptor or an antibody reactive against the peptide sequence LHAL (SEQ ID NO: 5). The human 5HT1B/1D receptor antagonist can be, for example, an anti-receptor antibody.